Group of nucleic acid molecules salmonella detection, nucleic acids, kit and use

ABSTRACT

The present invention relates to a nucleic acid molecule or molecules and to a process for the detection of bacteria of the Salmonella genus. The invention relates also to a test kit or test kits for carrying out the mentioned detection processes.

In the foodstuffs and pharmaceutical industries the contamination ofproducts by pathogenic microorganisms by way of the raw materials usedor during the production or packaging processes poses a major problem.Salmonellae are among the most serious pathogens transmitted to humansthrough foodstuffs. Since the detection and identification ofSalmonellae by conventional microbiological detection processes is verytime-consuming—at least five days are required for the increase inquantity and subsequent serotyping required by legal regulations (LMBG,FDA)—there is a great need for alternative rapid methods.

In recent years, a number of new methods have been developed for routineuse to detect microorganisms. These include immunological processesbased on the use of polyvalent or monoclonal antibodies and processes inwhich nucleic acid probes are used for detection by means ofhybridisation to organism-specific nucleic acids. Further methods thathave been described are those processes based on a specific nucleic acidamplification, with or without a subsequent confirmation reaction bynucleic acid hybridisation. Suitable processes for the amplification ofnucleic acids are, for example, polymerase chain reaction [PCR] [U.S.Pat. Nos. 4,683,195; 4,683,202; and 4,965,188], ligase chain reaction[WO Publication 89/09835], “self-sustained sequence replication” [EP 329822], the “transcription based amplification system” [EP 310 229] andthe Qβ RNA-replicase system [U.S. Pat. No. 4,957,858].

The mentioned nucleic-acid-based processes are so sensitive that, unlikeconventional microbiological processes, a lengthy increase in quantityof the microorganism to be detected from the sample to be investigatedis unnecessary. An investigation of the presence or absence of, forexample, Salmonellae is therefore generally concluded within one workingday when using the mentioned nucleic-acid-based processes.

Some nucleic acid sequences for detecting Salmonellae by polymerasechain reaction are known. A disadvantage is, however, that when usingthose nucleic acid sequences as primers in the polymerase chain reactionfalse positive results [WO 95/33854] or false negative results [WO92/01056; WO 95/00664; WO 92/01056; WO 93/04202] occur. In other cases,only an insufficient number of strains of all 7 Salmonellae subspecieshave been studied [WO 92/08805; WO 94/25597; DE 4337295], so that thusfar it is unclear whether the nucleic acid sequences in question aresuitable for detecting all Salmonella strains.

An advantage of, for example, the primers and probes described inInternational Patent Application WO 95/00664 is that they allow thehighly selective detection of bacteria of the Salmonella genus withoutthe occurrence of false positive results. A disadvantage when using theoligonucleotides according to WO 95/00664 in amplification processessuch as polymerase chain reaction is, however, the fact that none of thedescribed primer pairs enable detection of all the representatives ofthe 7 Salmonella subspecies. For example, when using the primersST11/ST15, a number of representatives of subspecies IIIa (subsp.arizonae) are not detected, and when using the primers ST11/ST14 anumber of representatives of subspecies I (subsp. enterica Serovar.Blockley) and of subspecies IIIa (subsp. arizonae) are not detected.

An aim of the invention described herein was to optimise the detectionprocesses described in WO 95/00664 by finding nucleic acid sequences theuse of which as primers and/or probes ensures as complete detection aspossible of all the representatives of the Salmonella genus.

According to an embodiment, the problem underlying the invention issolved by a set of nucleic acid molecules by means of which, in aprocess for the detection of representatives of Salmonella entericasubsp. enterica, salamae, arizonae, diarizonae, houtenae, bongori andindica, all the representatives of those subspecies can be detected, theset being obtainable by

(a) obtaining or deriving a first nucleic acid molecule (nucleic acidmolecule 1) in a manner known per se using a nucleic acid isolate of arepresentative of one of the mentioned Salmonella enterica subspecies,which first nucleic acid molecule is specifically suitable as primer orprobe for the detection of that representative or of further or allrepresentatives of that one Salmonella enterica subspecies and possiblyalso of representatives of further Salmonella enterica subspecies,

(b) obtaining or deriving a second nucleic acid molecule (nucleic acidmolecule 2) in a manner known per se using a nucleic acid isolate of adifferent representative of one of the mentioned Salmonella entericasubspecies, which second nucleic acid molecule is specifically suitableas primer or probe for the detection of that representative or offurther or all representatives of that different Salmonella entericasubspecies and possibly also of representatives of others of thementioned Salmonella enterica subspecies, and

(c) unless it is already possible to detect all the representatives ofthe mentioned Salmonella enterica subspecies using the nucleic acidmolecules obtainable according to (a) and (b), continuing to obtain orderive nucleic acid molecules according to (a) and/or (b) until all therepresentatives of the mentioned Salmonella enterica subspecies can bedetected using the obtained or derived set of nucleic acid molecules.

A derived nucleic acid molecule may be a nucleic acid molecule that canbe hybridised with the obtained nucleic acid molecule and thatpreferably has the same number of bases, possible hybridisationconditions being:

temperature≧25° C. and 1M NaCl concentration.

A derived nucleic acid molecule may be, for example, a nucleic acidmolecule the sequence of which has been determined by computer designand that has subsequently been manufactured and obtained by chemicalsynthesis.

The solution to the problem underlying the invention can also bedescribed as the provision of one or more nucleic acid molecule(s) Y (Z,. . . ), that(those) nucleic acid molecule(s) being characterised inthat the use of that(those) nucleic acid molecule(s)—in addition to theuse of a nucleic acid molecule (X)—in a process for the detection ofbacteria of the Salmonella genus enables the detection also ofSalmonella strains or Salmonella isolates that cannot be detected or canbe detected only with relatively low sensitivity using the nucleic acidmolecule (X).

The set of nucleic acid molecules according to the invention can becharacterised in that the nucleic acid isolates comprise or arephylogenetically conserved base sequences or regions of those basesequences. For the term “phylogenetically conserved base sequence”, see,for example, WO 95/00664 or Herder's Lexikon der Biochemie undMolekularbiologie, supplemented 1995, page 132, spectrum, production,etc.

The set of nucleic acid molecules according to the invention can becharacterised in that the individual nucleic acid molecules or some ofthe nucleic acid molecules hybridise to

(i) different phylogenetically conserved base sequences, or

(ii) one and the same phylogenetically conserved base sequence atnon-overlapping sequence regions, or

(iii) one and the same phylogenetically conserved base sequence atoverlapping sequence regions.

The set of nucleic acid molecules according to the invention or a set ofnucleic acid molecules according to the invention by means of which, ina process for the detection of representatives of Salmonella entericasubsp. enterica, salamae, arizonae, diarizonae, houtenae, bongori andindica, all the representatives of those subspecies can be detected, canbe characterised in that the set for an individual nucleic acidmolecule, for a number of its individual nucleic acid molecules or foreach of its individual nucleic acid molecules in each case comprises atleast one further nucleic acid molecule that, in a region of at least 10successive nucleotides of their nucleotide chain, corresponds to lessthan 100% but to at least 80% of the base sequence.

Such a set of nucleic acid molecules according to the invention can becharacterised in that the set for an individual nucleic acid molecule,for a number of its individual nucleic acid molecules or for each of itsindividual nucleic acid molecules in each case comprises at least onefurther nucleic acid molecule that, in a region of at least 10successive nucleotides of their nucleotide chain, differs from the otheror further nucleic acid molecule in precisely one base position.

A set of nucleic acid molecules according to the invention can becharacterised in that it comprises one or more, but not exclusively,nucleic acid molecules that are fragments of the SEQ ID NO 1 accordingto WO 95/00664 or of its complementary sequence.

A set of nucleic acid molecules according to the invention can also becharacterised in that the individual nucleic acid molecules hybridise tothe same strand of nucleic acid isolates of representatives ofSalmonella enterica subspecies that are being subjected to the processfor their detection.

The problem underlying the invention is also solved by a nucleic acidmolecule that belongs to a set of nucleic acid molecules according tothe invention or that can be used for such a set, the nucleic acidmolecule being characterised in that, in a region of at least 10successive nucleotides of its nucleotide chain, the sequence of thenucleic acid molecule corresponds exactly to a sequence region of atleast one representative of the mentioned Salmonella entericasubspecies, the sequence region comprising or being a phylogeneticallyconserved base sequence or a region of that base sequence.

Such a nucleic acid molecule according to the invention can becharacterised in that, in a region of at least 10 successive nucleotidesof its nucleotide chain, it is 100% or at least 80% identical to acorresponding number of successive nucleotides of one or more of thefollowing sequences or their complementary sequences:

ATGGATCAGAATACGCCCCG SEQ ID NO:1 ATGGATCAGAATACACCCCG SEQ ID NO:2CAGAATACGCCCCGTTCGGC SEQ ID NO:3 CAGAATACACCCCGTTCGGC SEQ ID NO:4CAGAATACGCCCCGTTCAGC SEQ ID NO:5 CAACCTAACTTCTGCGCCAG SEQ ID NO:6CAACCTAACTTCTGCACCAG SEQ ID NO:7 CAACCTAACCTCTGCGCCAG SEQ ID NO:8CAACCTAACTTCTGCGCCAG SEQ ID NO:9

The problem underlying the invention is also solved by a nucleic acidmolecule characterised in that, in respect of its sequence, it ishomologous to an above-characterised nucleic acid molecule according tothe invention and, in at least 10 successive nucleotides of itsnucleotide chain,

(i) is identical to an above-characterised nucleic acid moleculeaccording to the invention, or

(ii) differs from an above-characterised nucleic acid molecule accordingto the invention in not more than one nucleotide, or

(iii) differs from an above-characterised nucleic acid moleculeaccording to the invention in not more than two nucleotides.

A nucleic acid molecule according to the invention can be characterisedin that it is from 10 to 250 nucleotides long and preferably from 15 to30 nucleotides long.

A nucleic acid molecule according to the invention can also becharacterised in that it is single-stranded or has a complementarystrand.

A nucleic acid molecule according to the invention can also becharacterised in that it is present

(i) as DNA, or

(ii) as RNA corresponding to (i), or

(iii) as PNA, the nucleic acid molecule where appropriate having beenmodified or labelled in a manner known per se for analytical detectionprocesses, especially detection processes based on hybridisation and/oramplification.

A nucleic acid molecule according to the invention can also becharacterised in that it is a modified or labelled nucleic acid moleculein which up to 20% of the nucleotides of at least 10 successivenucleotides of its nucleotide chain are building blocks known per se asprobes and/or primers, especially nucleotides that do not occurnaturally in bacteria.

A nucleic acid molecule according to the invention can also becharacterised in that it is a modified or labelled or additionallymodified or labelled nucleic acid molecule that comprises, in a mannerknown per se for analytical detection processes, one or more radioactivegroups, coloured groups, fluorescent groups, groups for immobilisationon a solid phase, groups for an indirect or direct reaction, especiallyfor an enzymatic reaction, preferably using antibodies, antigens,enzymes and/or substances having an affinity for enzymes or enzymecomplexes, and/or other modifying or modified groups ofnucleic-acid-like structure that are known per se.

The problem underlying the invention is also solved by a kit foranalytical detection processes, especially for the detection of bacteriaof the Salmonella genus, that kit being characterised by

(i) a set of nucleic acid molecules according to the invention, or

(ii) one or more nucleic acid molecules according to the invention.

A kit according to the invention can thus comprise a set of nucleic acidmolecules according to the invention or one or more nucleic acidmolecules according to the invention, there additionally being providedthe other customary components for nucleic acid hybridisations ornucleic acid amplifications, for example a polymerase, a reversetranscriptase, a ligase or an RNA-polymerase, see, for example, WO95/00664.

A set of nucleic acid molecules of the kit according to the inventionwill preferably be produced synthetically in at least two separatesynthesis batches. The kit according to the invention preferably doesnot comprise any degenerate nucleic acid molecules.

Finally, the problem underlying the invention is solved by the use of aset of nucleic acid molecules according to the invention or of a kitaccording to the invention to detect the presence or absence of bacteriabelonging to a group of bacteria of the Salmonella genus, especially ofrepresentatives of the above-mentioned Salmonella enterica sub-species.

For the use according to the invention, nucleic acid hybridisationand/or nucleic acid amplification can be carried out.

As nucleic acid amplification, there can be carried out a polymerasechain reaction (PCR).

For the use according to the invention, differences between the genomicDNA and/or RNA of the bacteria to be detected and of the bacteria thatare not to be detected can be determined at at least one nucleotideposition in the region of a nucleic acid molecule according to theinvention and representatives of a group of bacteria of the Salmonellagenus can be detected, especially representatives of the mentionedSalmonella enterica subspecies.

To detect Salmonellae by means of nucleic acid hybridisation oramplification, Salmonella-specific oligonucleotides are used.Salmonella-specific oligonucleotides are nucleic acid molecules, from 10to 250 bases (preferably from 15 to 30 bases) long, the base sequence ofwhich is characteristic for Salmonellae: when using sucholigonucleotides as primers or probes—with suitable reactionconditions—hybridisation/amplification takes place only when DNA of theSalmonellae to be detected is present in the test sample, but not whenDNA of other bacteria is present.

As described below, given certain prerequisites non-specificoligonucleotides may also be used as primers or probes. Sucholigonucleotides enable hybridisation and/or amplification not only whenSalmonella-DNA is present in the sample but also in the presence of DNAof a bacterium or of a number of bacteria not belonging to theSalmonella genus.

Since, in highly conserved gene regions, substitutions (e.g. pointmutations) in the DNA can occur even in the case of very closely relatedbacteria, comprehensive DNA sequencing or specificity tests (e.g. bycarrying out PCR) are necessary to select suitable oligonucleotides.This applies equally to bacteria to be detected (i.e. Salmonellae) andto bacteria that are not to be detected (i.e. bacteria not belonging tothe Salmonella genus).

To detect Salmonellae, firstly nucleic acids, preferably genomic DNA,are released from the cells contained in a sample or bacterial cultureto be investigated. By means of nucleic acid hybridisation, the directdetection of Salmonella nucleic acids in the sample to be investigatedcan then be effected using the Salmonella-specific oligonucleotidesaccording to the invention as probe. Various processes known to theperson skilled in the art are suitable for that purpose, such as, forexample, “Southern blot” or “dot blot”.

Preference is given, however, above all on account of the relativelyhigh sensitivity, to indirect detection in which the DNA/RNA sequencessought are firstly amplified by means of the above-mentioned processesfor amplifying nucleic acids, preferably PCR. The amplification ofDNA/RNA is effected by using Salmonella-specific oligonucleotides. Inthat process specific amplification products are formed only whenSalmonella-DNA/RNA is present in the sample to be investigated. Thespecificity of the detection process can be increased by a subsequentdetection reaction using Salmonella-specific oligonucleotides as probes.It is also possible to use non-specific oligonucleotides as probes.

Alternatively, amplification can also be carried out in the presence ofone or more non-specific oligonucleotides, so that possibly also DNA/RNAof other microorganisms that are not to be detected may be amplified.Such an amplification process is generally less specific and shouldtherefore be backed up by a subsequent detection reaction usingSalmonella-specific oligonucleotides as probe.

Various processes by which amplification products formed in the indirectprocesses can be detected are known to the person skilled in the art.These include, inter alia, visualisation by means of gelelectrophoresis, the hybridisation of probes on immobilised reactionproducts [coupled to nylon or nitrocellulose filters (“Southern blots”)or, for example, on beads or microtitre plates] and the hybridisation ofthe reaction products on immobilised probes (e.g. “reverse dot blots” orbeads or microtitre plates coupled with probes).

A large number of different variants have been described by means ofwhich the described Salmonella-specific or non-specific oligonucleotidesfor use as probes and/or primers in direct or indirect detectionprocesses can be labelled or modified. They may comprise, for example,radioactive, coloured or fluorescent groups or groups that enableimmobilisation on a solid phase or groups that have been modified orthat modify in some other way, such as, for example, antibodies,antigens, enzymes or other substances having an affinity for enzymes orenzyme complexes. Probes and primers may be either naturally occurringor synthetically produced double- or single-stranded DNA or RNA ormodified forms of DNA or RNA, such as, for example, PNA (in thosemolecules the sugar units have been replaced by amino acids orpeptides). Individual nucleotides or a number of nucleotides of theprobes or primers may be replaced by analogous building blocks (such as,for example, nucleotides that do not naturally occur in the targetnucleic acid). In the case of the above-mentioned indirect detectionprocesses, the detection can be carried out also by means of aninternally labelled amplification product. That can be effected, forexample, by the integration of modified nucleoside triphosphates (e.g.coupled with digoxygenin or fluorescein) during the amplificationreaction.

Suitable Salmonella-specific oligonucleotides according to the inventionare nucleic acids, preferably from 15 to 30 bases long, that correspond,at least in a 10 base long sequence, to sequences 1 to 10 or to theircomplementary sequences. Relatively small differences (1 or 2 bases) inthat 10 base long sequence are possible without loss of the requisitespecificity in the amplification and/or hybridisation. The personskilled in the art will know that in the case of such relatively smalldifferences the reaction conditions need to be altered accordingly.

In order to enable complete detection of all the Salmonella strainsusing the DNA region outlined in WO 95/00664, comprehensive DNA sequenceanalyses were necessary. The sequence of that DNA region was determinedfrom 37 selected Salmonella strains of all 7 subspecies (for most of thestrains, the sequence of the DNA region between primers ST15 and ST11;this corresponds to position 1275 to 1654 of SEQ ID NO: 1 in WO95/00664). Possible experimental procedures will be known to the personskilled in the art and will not be described here in detail; a briefsummary of the results will be given. DNA of the selected Salmonellastrains was prepared by standard procedures and the relevant region wasamplified by PCR and subsequently sequenced. In the PCR and thesubsequent sequencing, the following primers were used for most of theSalmonella strains:

ST11: AGCCAACATTGCTAAATTGGCGCA (SEQ ID NO:l1) (see claim 3, WO 95/00664)

ST15: GGTAGAAATTCCCAGCGGGTACTG (SEQ ID NO:12) (see claim 3, WO 95/00664)

Since, however, no amplification, or only insufficient amplification,occurred with that primer pair with a number of strains of subspeciesIIIa, IV, V and VI, in those cases the following primers were used forthe PCR and sequencing:

ST11:AGCCAACCATTGCTAAATTGGCGCA (see claim 3, WO 95/00664)

ST14:TTTGCGACTATCAGGTTACCGTGG (SEQ ID NO:13) (see claim 3, WO 95/00664).

A comparison of the DNA sequences of all 37 Salmonella strains showedthat while it was as a whole a conserved DNA region, the degree ofconservation appeared at first glance to have only limited suitabilityfor deriving Salmonella-specific oligonucleotides. Even in the mosthighly conserved regions, base substitutions were observed in some ofthe sequenced strains. Interestingly, it was found that many of the basesubstitutions occur only within a subgroup and that the substitutionsare moreover generally conserved within that subgroup. This suggestedthe possibility of using more than two primers in the PCR in order toenable amplification also of those variants in which one or more basesubstitutions are present in the region of the primer binding sites. Asthe person skilled in the art will know, for that purpose there arecustomarily used degenerate primers or primers having deoxyinosin at thevariable sites. A number of degenerate oligonucleotides that werepotentially suitable as primers for the detection of all Salmonellaenterica subspecies were therefore deduced from the above-mentionedsequence comparison. It was found, however, that those degenerateprimers have only limited suitability for PCR detection since theyresult in an increase in the occurrence of non-specific reactionproducts, especially in the case of sequence regions of high complexity.Since the sensitivity of the PCR detection generally suffers from theoccurrence of such non-specific reaction products, a different procedurewas tried. “Complementing” primers were used in the PCR. In contrast todegenerate primers, in which all the possible combinations of theindividual base substitutions are represented in the primer mixture(number of primers=2^(x)×3^(Y)×4^(z) where x, y and z are the number ofpositions at which two, three or four different bases are observed inthe region of the primer binding site), in such complementing primersonly the actually occurring sequences are present. The advantage overdegenerate primers lies in the lesser complexity of the primer mixtureaccording to the invention, as a result of which the probability thatnon-specific amplification products will be formed is markedly reduced.As has been shown in a number of experiments, this is especiallyadvantageous in PCR detection using samples having a high content of“non-specific” DNA (DNA that does not come from bacteria to be detected)since, otherwise, the sensitivity of the detection may be radicallyreduced.

A major advantage when using complementing oligonucleotides/primers liesin the possibility of optimising existing detection processes. Forexample, it is possible that individual false negative results can beeliminated by additionally using in the PCR and/or hybridisationreaction oligonucleotides comprising the sequence of the previouslyundetected strains.

The DNA sequence comparison yielded a number of relatively short DNAregions that appeared to be potentially suitable for the strategydescribed (use of in total ≧3 primers in the PCR) for optimising theSalmonella detection process. The following Example is given by way ofclarification.

EXAMPLE 1 Detection of Salmonella Strains of all 7 Subspecies byPolymerase Chain Reaction

The following 3 sections of the sequenced DNA region are to serve asexamples for the sequence variations observed:

Section I (position 1336 to 1355 of SEQ ID NO: 1 in WO 95/00664)

ATGGATCAGAATACGCCCCG SEQ ID NO:1 ATGGATCAGAATACACCCCG SEQ ID NO:2

Section II (position 1342 to 1361 of SEQ ID NO: 1 in WO 95/00664)

CAGAATACGCCCCGTTCGGC SEQ ID NO:3 CAGAATACACCCCGTTCGGC SEQ ID NO:4CAGAATACGCCCCGTTCAGC SEQ ID NO:5

Section III (complementary to position 1483 to 1502 of SEQ ID NO: 1 inWO 95/00664)

CAACCTAACTTCTGCGCCAG SEQ ID NO:6 CAACCTAACTTCTGCACCAG SEQ ID NO:7CAACCTAACCTCTGCGCCAG SEQ ID NO:8 CAACCTAACTTCTGCGGCAG SEQ ID NO:9CAACCTAACTTCTGCGGCAG SEQ ID NO:10

To test whether those sequence sections are suitable for detecting allthe Salmonella strains of the 7 subspecies, the oligonucleotides Sa 1 to10 were used in the PCR in the following combinations:

Primer combination 1 Sa1/Sa2 (each in a final concentration of 0.2 μM)Sa6/Sa7/Sa8/Sa9/Sa10 (each in a final concentration of 0.08 μM) Primercombination 2 Sa3/Sa4/Sa5 (each in a final concentration of 0.13 μM)Sa6/Sa7/Sa8/Sa9/Sa10 (each in a final concentration of 0.08 μM)

DNA was isolated by standard processes from pure cultures of theSalmonella strains listed in Table 1a. Approximately from 10 to 100 ngof each of those DNA preparations was then used in the PCR in thepresence of primer combination 1 or primer combination 2, 200 μM ofdNTP's (Boehringer Mannheim), 1.5 mM MgCl₂, 16 mM (NH₄)₂SO₄, 67 mMTris/HCl (pH 8.8), 0.01% Tween 20 and 0.03 U/μl Taq-polymerase(Biomaster). The PCR was carried out in a Perkin-Elmer 9600 thermocyclerhaving the following thermoprofile:

initial denaturing 95° C.  5 min amplification (35 cycles) 95° C. 30 sec63° C. 90 sec final synthesis 72° C.  5 min

After the end of the PCR reaction, the amplification products wereseparated by means of agarose gel electrophoresis and visualised bystaining with ethidium bromide. The expected product of 167 bp length(primer combination 1) or of 161 bp length (primer combination 2 wasobserved in all cases in which DNA of strains of the Salmonella genuswas present (compare Table 1a), but not in the presence of DNA of othertested bacteria (compare Table 1b). After the end of the run, the DNAcontained in the gels was transferred by standard methods to nylonfilters and hybridised with the oligonucleotide ST14(TTTGCGACTATCAGGTTACCGTGG (SEQ ID NO:13) (see claim 3, WO 95/00664))labelled at the 5′ end with digoxygenin to test the base specificityespecially sensitively. Hybridisation was effected in 5×SSC, 2% blockingreagent, 0.1% lauryl sarcosine, 0.02% SDS and 5 pmol/ml of probe for 4hours at 60° C. Washing was carried out in 2×SSC, 0.1% SDS for 2×15minutes at 60° C. Detection was carried out according to standardmethods using anti-digoxygenin/alkaline phosphate conjugates in thepresence of 5-bromo-4-chloro-3-indolyl phosphate and 4-nitro-bluetetrazolium chloride (Boehringer Mannheim).

A band was observed on the filters only in those cases in which a bandhad previously been visible on the agarose gel (see Table 1a). Thus, thepresence of the 296 tested Salmonella strains from each of the 7subspecies was detected both by PCR and by hybridisation. A positivesignal was obtained for each of those strains with primer combination 1,primer combination 2 and, in the subsequent confirmation reaction, byhybridisation with the probe ST14. By contrast, none of the testedbacterial strains not belonging to that genus was detected using thissystem.

TABLE 1a Positive Salmonella strains in PCR amplification using the twoprimer combinations 1 or 2 and in the subsequent hybridisation using theoligonucleotide ST14. No. Subspecies Serogroup Serovar. S. enterica BAbony subsp. Abortusovis Enterica Africana Agona Agona, lactose +Arechavaleta Brandenburg Bredeney Chester Coeln Derby O: 5 − DuisburgDuisburg, monophase Heidelberg Heidelberg, O5 − I 4, 12: d: − I 4, 12:−: − I 9, 12: 1, v: − Indiana Kiambu Kunduchi Paratyphi B Paratyphi B H1, 2 negative Paratyphi B O5 − Reading Saintpaul O5 − Saintpaul SandiegoSchleisheim Schwarzengrund Stanley Stanleyville Typhimurium Typhimurium4: i: 1, 2 (O: 5−) Typhimurium O: 5 − Typhimurium, TA 1535 Typhimurium,TA 1537 Typhimurium, TA 1538 Typhimurium, TA 97 Typhimurium, TA 98Typhimurium, TA 100 C₁ Augustenborg Bareilly Braenderup CholeraesuisCholeraesuis var. Decatur Choleraesuis var. Kunzendorf Colindale ConcordInfantis Isangi Lille Livingstone Mbandaka Mikawasima Montevideo OhioOranienburg Oslo Richmond (2 isolates tested) Rissen Singapore TennesseeThompson (2 isolates tested) Virchow I 6, 7: −: − (2 isolates tested) C₂C₃ Albany (2 isolates tested) Altona Apeyeme Bardo BlockleyBovismorbificans Charlottenburg Cottbus Emek Ferruch Glostrup GoldcoastHaardt Hadar Kentucky Litchfield Manchester Manhattan Molade MunchenNewport Takoradi I 6, 8: −: − I 8, 20: −: − D₁ Dublin Durban EnteritidisEnteritidis plasmid phage 37MD EnteritidiS PT 4/6 Enteritidis, phageGallinarum Gallinarum-Pullorum Israel Javiana Kapemba Napoli PanamaPullorum I 9, 12: −: − D₂ Plymoth E₁ Amager Amsterdam O: −, 15+, 34+Anatum Anatum O 15+ Anatum O: 10−, O: 15+ Birmingham Butantan FalkenseeGive Lexington London Meleagridis Munster Munster, O: 10−, 15+ OrionOrion O: 10 −, 15+ 34+ Sinstorf Stockholm Uganda (2 isolates tested)Vejle (2 isolates tested) Weltevreden Westhampton Zanzibar I 3, 10: −: 6(monophase) I 10: −: 1, 6 E₄ Abaetuba Aberdeen Cannstatt LlandoffSenftenberg, delayed Lac. + I 1, 3, 19, : −: − F Chandans (2 isolatestested) Kisarawe Krefeld Liverpool Rubislaw Senftenberg Solt TelashomerG Grumpensis Havana Idikan Kedougou Poona Putten Worthington I 13, 23, :− H Caracas Charity Lindern Onderstepoort Sundsvall GaminaraHvittingfoss Malstatt Saphra J Bonames K Cerro L Minnesota (2 isolatestested) Ruiru M Cotham Guildford Ilala Loeben Mundonobo Nima PatiencePomona Taunton Wedding N Aqua Morningside Urbana O Adelaide AlachuaEaling Haga Monschaui P Lansing Roan (2 isolates tested) Shettfield QKokomelemle R Johannesburg S Waycross (2 isolates tested) T Waral UThetford V Koketime (2 isolates tested) Lawra W Suelldorf X I 47, z₄,z₂₃: (monophase) Mountpleasant S. enterica B II 4, 12: a: − subsp. C₁ II6, 7: d: 1, 7 salamae F II 11: g, m, s, t: z₃₉ I II 16: g, m, s, t: − JII 17: c: z₃₉ II 17: b: e, n, x, z₁₅ L II 21: z₁₀: − P II 38: d: 1, 5 RII 1, 40: z₄₂: 1, 5, 7 S II 41: z₁₀, 1, 2 T II 42: r: − (3 isolatestested) X II 47: a: 1, 5 (2 isolates tested) II 47: b: 1, 5 (2 isolatestested) II 47: b: z₆ Z II 50: b: z₆ (5 isolates tested) O: 58 II 58: 1,z₁₃ , z₂₈: z₆ S. enterica J IIIa 17: z₄, z_(32: −) subsp. K IIIa 18: z₄,z₂₃: − arizonae P IIIa 38: 1, v: − R IIIa 40: z₄, z₂₄: − S IIIa 41: z₄,z₂₃: − U IIIa 43: g, z₅₁: − V IIIa 44: z₄, z₃₂: − IIIa 44: z₄₁, z₂₃: − YIIIa 48: (1): − IIIa 48: g, z₅₁ : − IIIa 48: z₃₆: − IIIa 48: z₄, z₂₃: −Z IIIa 50: z₄, z₂₄: − O: 51 IIIa 51: z₄, z₂₃: − IIIa 51: g, z₅₁: − O: 53IIIa 53: z₄, z₂₃, z₃₂: − IIIa 53: z₂₉: − O: 62 IIIa 62: z₃₆: − O: 63IIIa 63: g, z₅₁: − S. enterica D₁ IIIb 1, 9, 12: y: z₃₉ subsp. I IIIb16: k: − diarizonae J IIIb 17: z₁₀, e, n, x, z₁₅ O IIIb 35: k: e, n, z₁₅P IIIb 38: 1, v: z₅₃ IIIb 38: 1, v: z₅₄ T IIIb 42: k: z₃₅ X IIIb 47: b:z₆ IIIb 47: k: z₃₅ IIIb 47: r: z₅₃ IIIb 47: −: − Y IIIb 48: (k): z₅₃ ZIIIb 50: k: z IIIb 50: r: z O: 53 IIIb 53: 1, k: z O: 60 IIIb 60: z₅₂:z₅₃ O: 61 IIIb 61: 1: z IIIb 61: 1, v: 1, 5, 7 IIIb 61: 1, v: 1, 5, 7:(z₅₇) IIIb 61: r: z₅₃ S. enterica F IV 11: z₄, z₂₃: − subsp. I IV 16:z₄, z₃₂: − houtenae I IV 16: z₄, z₃₂: − J IV 17: z₂₉: − K IV 18: z₃₆,z₃₈: − L IV 21: g, z₅₁: − U IV 43: z₄, z₂₃: − IV 43: z₄, z₃₂: − V TV 44:z₄, z₃₂: − Y IV 48: z₂₉: − Z IV 50: z₄, z₂₃: − S. enterica R V 40: z₃₅:− subsp. V 40: z₈₁: − bongori V V 44: d: − V 44 : z₃₉: − Y V 48 : z₃₅: −S. enterica S VI 41: b: 1, 7 subsp. W VI 45: a: e, n, x, (z₁₇) indica YVI 48: z₁₀: 1, 5 VI 48: z₄₁: − VI 1, v: z₆₇ Reference source: bgVVBerlin, (Robert von Ostertag Institut, Marienfelde) and Institut furMikrobiologie und Hygiene, Hamburg (Prof. Aleksic). The strains bearingthe letters TA are strains used for the Ames Test (Maron, D. M. & Ames,B. N., Mutation Research 113, 173—215 (1983)).

TABLE 1b Negative strains of non-Salmonella species in PCR amplificationusing the two primer combinations 1 or 2 and the subsequenthybridisation using the oligonucleotide ST 14 No. Species OriginBacillus subtilis ATCC 6051 Citrobacter freundii DSM 30040 Clostridiumbifermentans DSM 630 Enterobacter agglomerans IfGB 0202 Enterobactercloacae DSM 30054 Erwinia carotovora DSM 30168 Escherichia coli ATCC8739 Hafnia alvei IfGB 0101 Klebsiella oxytoca DSM 5175 Klebsiellapneumoniae ATCC 13883 Klebsiella oxytoca DSM 5175 Lactobacillus spec.ATCC 20182 Listeria monocytogenes ATCC 19118 Pediococcus domnatus IfGB0101 Proteus vulgaris DSM 2041 Pseudomonsas fluorescens DSM 6290Serratia marcescens IfGB 0101 Shigella flexneri DSM 4782 Staphylococcusaureus ATCC 6538 Yersinia enterocolitica DSM 4780

13 1 20 DNA Salmonella enterica 1 atggatcaga atacgccccg 20 2 20 DNASalmonella enterica 2 atggatcaga atacaccccg 20 3 20 DNA Salmonellaenterica 3 cagaatacgc cccgttcggc 20 4 20 DNA Salmonella enterica 4cagaatacac cccgttcggc 20 5 20 DNA Salmonella enterica 5 cagaatacgccccgttcagc 20 6 20 DNA Salmonella enterica 6 caacctaact tctgcgccag 20 720 DNA Salmonella enterica 7 caacctaact tctgcaccag 20 8 20 DNASalmonella enterica 8 caacctaacc tctgcgccag 20 9 20 DNA Salmonellaenterica 9 caacctaact tctgcgccag 20 10 20 DNA Salmonella enterica 10cagcctaact tctgcgccag 20 11 25 DNA Salmonella enterica 11 agccaaccattgctaaattg gcgca 25 12 24 DNA Salmonella enterica 12 ggtagaaattcccagcgggt actg 24 13 24 DNA Salmonella enterica 13 tttgcgactatcaggttacc gtgg 24

What is claimed is:
 1. A method of detecting the presence or absence ofa subspecies of bacterium Salmonella enterica, comprising the steps of:(a) providing at least one isolated nucleic acid molecule selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ 1DNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9 and SEQ ID NO: 10 and the complement of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10; (b) contacting the atleast one isolated nucleic acid molecule of (a) with a test samplecontaining nucleic acids; (c) hybridizing and/or amplifying the nucleicacid molecules of the test sample with the at least one isolated nucleicacid molecule of (a); and (d) detecting nucleic acid hybrids formedbetween the nucleic acid molecules of the test sample and the at leastone nucleic acid molecule of (a) and/or detecting amplified nucleic acidmolecules of the test sample, thereby determining the presence orabsence of a subspecies of the bacterium Salmonella enterica in the testsample.
 2. The method of claim 1, wherein the amplifying of the nucleicacid molecules of the test sample is carried out by a polymerase chainreaction (PCR).
 3. The method of claim 1, wherein the subspecies ofSalmonella enterica is selected from the group consisting of Salmonellaenterica subspecies enterica, salamae, arizonae, diarizonae, houtenae,bongori and indica.
 4. A kit comprising: (i) one or more isolatednucleic acid molecules selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 and thecomplement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 andSEQ ID NO: 10, and (ii) optionally substances for analytical detectionprocesses.
 5. The kit of claim 4, wherein at least one isolated nucleicacid molecule is modified or labelled with a group selected from thegroup consisting of a radioactive group, a colored group, a fluorescentgroup, a group for immobilisation on a solid phase and a group allowingan indirect or direct enzyme reaction.
 6. The kit of claim 5, whereinthe group allowing an enzyme reaction is selected from the groupconsisting of antibodies, antigens, enzymes, substances having anaffinity for enzymes and substances having an affinity for enzymecomplexes.
 7. The kit of claim 4, wherein the isolated nucleic acidmolecule is single stranded.
 8. The kit of claim 4, wherein the isolatednucleic acid molecule is double stranded.
 9. The kit of claim 4, whereinthe isolated nucleic acid molecules are produced synthetically and in atleast two separate synthesis batches.